West Nile Virus Lineage 1 in Italy: Newly Introduced or a Re-Occurrence of a Previously Circulating Strain?

In Italy, West Nile virus (WNV) appeared for the first time in the Tuscany region in 1998. After 10 years of absence, it re-appeared in the areas surrounding the Po River delta, affecting eight provinces in three regions. Thereafter, WNV epidemics caused by genetically divergent isolates have been documented every year in the country. Since 2018, only WNV Lineage 2 has been reported in the Italian territory. In October 2020, WNV Lineage 1 (WNV-L1) re-emerged in Italy, in the Campania region. This is the first occurrence of WNV-L1 detection in the Italian territory since 2017. WNV was detected in the internal organs of a goshawk (Accipiter gentilis) and a kestrel (Falco tinnunculus). The RNA extracted in the goshawk tissue samples was sequenced, and a Bayesian phylogenetic analysis was performed by a maximum-likelihood tree. Genome analysis, conducted on the goshawk WNV complete genome sequence, indicates that the strain belongs to the WNV-L1 Western-Mediterranean (WMed) cluster. Moreover, a close phylogenetic similarity is observed between the goshawk strain, the 2008–2011 group of Italian sequences, and European strains belonging to the Wmed cluster. Our results evidence the possibility of both a new re-introduction or unnoticed silent circulation in Italy, and the strong importance of keeping the WNV surveillance system in the Italian territory active.


Introduction
West Nile virus (WNV) is a mosquito borne single-stranded RNA virus, a member of the Japanese encephalitis (JE) serocomplex belonging to the genus Flavivirus within the Flaviviridae family [1]. WNV is maintained in nature through an endemic cycle which involves mosquitoes (Diptera; Culicidae) as vectors, and birds as reservoir hosts [2]. Humans and horses are considered "dead-end" hosts: they may develop disease; however, they are not able to infect vectors, and maintain the virus in the environment [3]. To date, eight different lineages of WNV have been described [4]. Lineages 1 and 2, often associated with cases of encephalitis in humans and horses, are by far those most widespread in Europe and the Mediterranean basin [5][6][7][8].
In Europe, WNV lineage 1 (WNV-L1) circulation was first evidenced in the 1960s in France, Portugal, and Cyprus [8][9][10]. Thirty years later, cases associated to WNV-L1 infection were reported in humans and horses in North African, Western, and Eastern European countries [8]. The WNV-L1 strains responsible for the Morocco (1996), Italy (1998), Israel (1998), and France (2000) human and horse cases [11][12][13][14][15][16] grouped into the (Trentola Ducenta municipality 40.976368, 14.1664) provinces, respectively ( Figure 1). The two birds, immediately transferred to the Regional Center for Wild Animals (CRAS) for rescue operations, died 48 h after the transfer. In detail, at the time of admission at the clinic, the kestrel was in a comatose state showing mydriasis. The radiography also evidenced the fracture of the tibia, and the presence of two bullet fragments. At the time of acceptance, the goshawk showed head injury, head tilt, and right lower limb ataxia. Necropsies were carried out, and selected tissues (heart, kidney, spleen, and brain) were collected, pooled, and homogenized in a sterile phosphate-buffered saline (PBS). Viral RNA was extracted from 200 µL supernatants using Qiasymphony ® DSP automatic instrumentation (Germantown, MD, USA) according to the manufacturer's instructions. Quantitative reverse transcription polymerase chain reactions (qRT PCR) to detect WNV-L1 and/or -L2 RNA was performed at the U.O.C. Virology of IZSM as described by Del Amo and colleagues [60]. The samples were sent to the National Reference Centre for Foreign Animal Diseases (CESME) at the Istituto Zooprofilattico Sperimentale of Abruzzo and Molise in Teramo (IZSAM) for WNV confirmation and further analysis.

Virus Strain and Laboratory Tests (IZSAM)
Virus RNA of the two samples' homogenates was extracted at IZSAM by using the MagMAX CORE Nucleic Acid Purification KIT (Applied Biosystem, Thermo Fisher Scientific, Life Technologies Corporation, TX, USA) according to the manufacturer's instructions. The virus RNA was tested by two qRT-PCR: (i) a 1-step RT-PCR assay for the simultaneous detection of WNV-L1 and 2 strains, by using the QuantiTect Probe RT-PCR Kit (QIAGEN) [60]; and (ii) an RT-PCR assay for detection of all known lineages of West Nile virus [61], by using the Superscript III Platinum OneStep qRT-PCR System (Invitrogen). The RNA detected in the goshawk tissue samples was further fully sequenced by using next generation sequencing (NGS) technology. Briefly, total RNA was treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) at 37 • C for 20 min, and then purified by an RNA Clean & Concentrator™-5 Kit (Zymo Research, Irvine, CA, USA). The purified RNA was used for the assessment of sequencing independent single primer amplification protocol (SISPA) [62,63]. In detail, viral RNA underwent cDNA synthesis using 200 units of the SuperScript ® IV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA), in the presence of 5X SSIV Buffer, 50 µM of the random hexamer FR26RV-N 5 -GCCGGAGCTCTGCAGATATCNNNNNN-3 , 10 mM of dNTPs mix, 100 mM of DTT, and 40U of RNAse OUT RNase inhibitor (Thermo Fisher Scientific, Waltham, MA). The reaction was incubated at 23 • C for 10 min, and 50 • C for 50 min. After an inactivation step at 80 • C for 10 min, 12.5 Units of 3 -5 Klenow Polymerase (New England Biolabs, Ipswich, MA, USA) were directly added to the reaction to perform the second strand cDNA synthesis. The incubation was carried out at 37 • C for 1 h, and 75 • C for 10 min. Next, 5 µL of ds cDNA were added to 45 µL of PCR master mix containing 5X Q5 Reaction Buffer, 10 mM of dNTPs, 40 µM of the random primer FR20 Rv 5 -GCCGGAGCTCTGCAGATATC-3 , Q5 ® High Fidelity DNA polymerase (NEB, New England Biolabs, Ipswich, MA, USA), and Q5 High Enhancer [64]. The reaction was incubated at 98 • C for 10 s, 65 • C for 30 s, 72 • C for 3 min, and 72 • C for 2 min. The PCR product was purified using the Molecular Biology Kit BioBasic (Biobasic inc., Markham, ON, Canada), and then quantified by using the Qubit ® DNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The sample was diluted to obtain a concentration of 100-500 ng, and used for library preparation by using the Illumina DNA Prep kit (Illumina Inc., San Diego, CA, USA) according to the manufacturer's protocol. Deep sequencing was performed on the NextSeq 500 (Illumina Inc., San Diego, CA, USA) using the NextSeq 500/550 Mid Output Reagent Cartridge v2, 300 cycles, and standard 150 bp paired end reads. FASTQ files were generated using NextSeq Reporter (Illumina). The sequencing run delivered 300 Mb of sequence data. The reads obtained were trimmed using a Trimmomatic script (Trimmomatic v0.36) to remove low quality and short reads [65]. Furthermore, the reads were quality controlled by using FastQC v0.11.5 [63,66]. The resulting 2,149,990 reads were de novo assembled using SPADES v3.11.1 [67]. Based on genome assemblies, a de novo filtering for the scaffolds with a minimum length of 200 nucleotides, and a matching for the best reference for each assembly using ABRicate was carried out [63]. Finally, a mapping with the references found in the previous step using Bowtie2 (v.2.1.0) was performed [68]. The length of the final assembly (GenBank accession number MW627239) was of 10,990 bp, and it showed 98.33% nucleotide identity to the reference sequence FJ483548.
The Bayesian phylogenetic analysis was performed through Bayesian Inference (BI) using a general time-reversible with gamma-distributed rate variation, a gamma category count of 4, and an invariant sites model (GTR + Γ + I), as selected by Akaike's information criterion (AICc) in jModelTest 0.1 [69]. A Bayesian MCMC approach using BEAST with JRE v2.6.3 was then employed. Ten independent MCMC runs with up to 100 million generations were performed to ensure the convergence of estimates. Tracer v.1.7.1 (available at http://beast.bio.ed.ac.uk/Tracer, accessed on 12 August 2021) was used to ensure convergence during MCMC by reaching effective sample sizes greater than 100. Trees were summarized in a maximum clade-credibility tree with common ancestor heights after a 10% burn-in [69] using TreeAnnotator v.2.6.3. A maximum likelihood tree was estimated using FigTree v1.4.4 [70] after identical alignment and curating methods. FigTree was run using the GTR + Γ + I nucleotide model with 2000 Γ-rate categories, exhaustive search settings, with 5000 bootstrap replications using the Shimodaira-Hasegawa (SH) test. All sequence alignments referred to in this manuscript can be found in Supplementary Materials.

WNV-L1 Detection
WNV-L1 was detected and confirmed in both the kestrel and the goshawk organs (heart, kidney, spleen, and brain pooled together in PBS).

Phylogenetic Tree Inferred with Maximum-Likelihood Analysis
Phylogenetic inference of WNV using a maximum-likelihood tree is shown in Figures 2 and 3. All Shimodaira-Hasegawa values are displayed at respective nodes.
A close similarity is observed between the MW627239 and the 2008-2011 groups of sequences clustered separately into the WMed subtype ( Figure 3). Phylogenetically, a similarity is also observed between the Italian WNV-L1 (2008,2011,2020) and the viral strains circulating in Europe in the recent past (Spain 2010, France 2015), although they are less temporally close than the Italian strains [71] [7,52,[73][74][75]. Results are shown in Table 2.
Using Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 12 August 2021), a nucleotide and amino acid pairwise identity analysis was conducted among MW627239 and the Italian, European, and American sequences listed above, as shown in Table 3. Among the Italian representative sequences belonging to the WNV-1 WMed single monophyletic group in the maximum-likelihood tree, the average nucleotide and amino acid pairwise identity was evidenced to be 98.13% (s.d. = 0.36) and 99.73 % (s.d = 0.07), respectively.
Results evidence a close genetic relatedness of the WNV-L1 strain that re-emerged in the Campania region in 2020, and the Italian and European strains belonging to the WNV-L1 WMed sub-cluster. Maximum likelihood phylogenetic tree of the WNV complete and partial genome sequences analyzed in this study. Violet, red, and green triangles represent the Mediterranean-Eastern European-Kenyan subtype, the Western Mediterranean subtype, and the Israeli-American subtype of WNV sequences, respectively. The Koutango virus strain EU082200 has been chosen as outgroup. The tree with the highest log-likelihood is shown. The Bayesian phylogenetic analysis was performed through Bayesian Inference (BI) using a general time-reversible with gamma-distributed rate variation, a gamma category count of 4, and an invariant sites model (GTR + Γ + I). The evolutionary distances were computed using the optimal GTR + Γ + I model, with 2000 Γ-rate categories and 5000 bootstrap replications using the Shimodaira-Hasegawa (SH) test. The percentage of successful bootstrap replicate (n5000) is indicated at nodes. A Bayesian MCMC approach using BEAST with JRE v2.6.3 was employed. Ten independent MCMC runs with up to 100 million generations were performed to ensure the convergence of estimates. Tracer v.1.7.1 was used to ensure convergence during MCMC by reaching effective sample sizes greater than 100. Trees were summarized in a maximum clade-credibility tree with common ancestor heights after a 10% burn-in using TreeAnnotator v2.6.3.

Figure 3.
Maximum likelihood phylogenetic tree of the WNV complete and partial genome sequences analyzed in this study. A detailed version of the phylogenetic tree is shown, showing all the Western Mediterranean WNV sequences. GenBank accession numbers are indicated for each strain, with country, lineage, and year of isolation. The WNV-L1 strain TE_362447_2020, obtained from the goshawk found in the Campania region in October 2020, is highlighted in red. The Koutango virus strain EU082200 has been chosen as outgroup. The tree with the highest log-likelihood is shown. The Bayesian phylogenetic analysis was performed through Bayesian Inference using a general time-reversible with gamma-distributed rate variation, a gamma category count of 4, and an invariant sites model (GTR + Γ + I). The evolutionary distances were computed using the optimal GTR + Γ + I model, with 2000 Γ-rate categories and 5000 bootstrap replications using the Shimodaira-Hasegawa (SH) test. The percentage of successful bootstrap replicate (n5000) is indicated at nodes. A Bayesian MCMC approach using BEAST with JRE v2.6.3 was employed. Ten independent MCMC runs with up to 100 million generations were performed to ensure the convergence of estimates. Tracer v.1.7.1 was used to ensure convergence during MCMC by reaching effective sample sizes greater than 100. Trees were summarized in a maximum clade-credibility tree with common ancestor heights after a 10% burn-in using TreeAnnotator v2.6.3.  I  I  I  I  I  I  I  I  I  I  I   E T  T  T  T  T  T  T  T  T  T  T   88  P  P  P  S  P  P  P  P  P  P  P   126  T  T  T  T  T  T  T  T  I  T  T   153  G  G  G  G  G  G  G  G  G  G  G   159  I  I  I  I  I  I  I  I  V  I  I   278  T  T  T  T  T  T  T  T  T  T  T Y   45  I  I  I  I  I  I  I  I  I  I  I  Table 2. Cont.  T  T   356  I  I  I  I  I  I  I  I  T  I  I M   526  T  T  T  T  T  T  T  T  T  T  T   653  F  F  F  F  F  F  F  F  F  F  F   681  T  T  T  T  T  T  T  T  T  T  T  Table 3. Nucleotide versus amino acid similarities of representative WNV Italian, European, and American strains. Pairwise identity analyses have been conducted among MW627239 and other WNV L-1 Italian, European, and American sequences, using Blast. Among the Italian representative sequences belonging to the WNV-1 WMed single monophyletic group in the maximum-likelihood tree, the average nucleotide and amino acid pairwise identity was evidenced to be 98.13% (s.d. = 0.36) and 99.73% (s.d = 0.07), respectively.

Discussion
This paper reports the first evidence of WNV-L1 strain circulation in the Campania region, Italy. The strain was detected in October 2020 in two wild birds found moribund in nearby areas, only a few days apart. It was the first detection of WNV-L1 after several years. The last WNV-L1 strain circulation evidenced in Italy dates back to 2017, when it was found in a pool of mosquitoes from Northern Italy (https://westnile.izs.it/j6_wnd/ wndItalia;jsessionid=D0C9EB639E7C322D0EFC34ECEB8E4D8E, 14 September 2021).
The last evidence of WNV-L1 circulation in an area most nearby the Campania region dates back to 2016 when the circulation of a WNV-L1 strain was responsible for the death of several wild birds in Sardinia [31]. The first question which clearly came to mind when tackling this finding was: "was this strain the result of a new introduction or was it just a re-occurrence of a strain already circulating?".
The first assumption supposes the virus probably extinguished and reintroduced through migratory birds. The species where the strains were detected did not help in clearing the question. Goshawks and kestrels can in fact be considered either migrant or resident birds. Unfortunately, it was not possible to check the carcasses, and eventually make out the bird behavior from wing, claw, and feather characteristics [74][75][76]. In support of the re-introduction of WNV-L1 is the lack of detection for consecutive years by the national surveillance program, which has been in place since 2002, and has been drawn to detect virus circulation early. In the same way, a phylogenetic relationship (%), and nucleotide and amino acid similarities are observed between the new sequence, MW627239, and some European sequences obtained in the past (Spain 2010, France 2015) (Figure 3, Tables 2 and 3). This might suggest a possible viral circulation in the Mediterranean followed, by a re-introduction in Italy in 2020.
Still on the phylogenic tree, however, the new strain MW627239 shows high nucleotide sequence identity (%) with the 2008-2011 Italian sub-clusters of the WMed single monophyletic group (Figure 3). In this respect, the second hypothesis, the WNV-L1 re-occurrence, seems to be the most credible scenario. Even if, in all these years, WNV has repeatedly proven its ability to overwinter and become endemic in many Italian regions, silence periods are factually not unusual for WNV-L1 [53,71]. After its first occurrence in the Tuscany region in 1998, WNV re-appeared in the areas surrounding the Po River delta after 10 years [55]. Similarly, in Sardinia, WNV-L1 was not detected for 3 consecutive years between 2011 and 2015 (https://westnile.izs.it/j6_wnd/wndItalia;jsessionid=D0C9EB639 E7C322D0EFC34ECEB8E4D8E, 14 September 2021). In this particular case, the WNV-L1 presence was accidentally uncovered in two wild birds when they were in critical conditions. The presence of clinical signs in birds would indeed facilitate the discovery of virus circulation on many occasions, as observed in several WNV epidemics of the early 2000s [16,76,77]. Even though the majority of the WNV infections in birds are usually mild or asymptomatic [78], some species, such as birds of prey, jays, and crows, are highly susceptible, and can develop severe and even fatal encephalitis [78][79][80]. Clinical symptoms associated to WNV-L1 infection have been mainly reported in the orders of Passeriformes (corvids, blue jays, magpies) and Falconiformes (birds of prey) [81][82][83]. Fatal infections have been described in European eagles in Spain [84,85], and geese and poultry in Hungary [24]. The two WNV-L1 infected birds found in Campania were in critical conditions. Though the comatose state of the kestrel was likely the consequence of a gunshot wound, the origin of the clinical picture observed in the goshawks is not easy to assess. Head injury, head tilt, and right lower limb ataxia are definitely signs of neurologic pathology. However, whether these symptoms were a consequence of the head injury or vice versa couldn't be determined. In nature, goshawks have been shown to be highly susceptible to WNV infection, probably because of their predatory habits [81]. Oral transmission of WNV by feeding on infected prey has been described, and is believed to be an important route of transmission in birds of prey [79,83,86]. Both WNV-L1 and L2 experimental infections conducted in American kestrels (Falco sparverius), golden eagles (Aquila chrysaetos), red-tailed hawks (Buteo jamaicensis), barn owls (Tyto alba), and great horned owls (Bubo virginianus) showed high level of viraemia, and important clinical symptoms, such as lethargy, inappetence, body weight loss, and muscle tremor [82,87,88]. However, looking back over the past 13 years of circulation in Italy, WNV-L1 seems to be unable to cause important clinical manifestations and deaths in birds [52].
The virulence of WNV depends on several factors related to the pathogen, hosts, and their interaction [89,90]. Concerning the pathogen, changes in the amino acid positions may significantly influence the WNV strain virulence [7]. Multiple genetic variations correlated with increased or decreased pathogenicity have been highlighted in genetic and phenotypic studies of WNV mutants [74]. Among the WNV genes, the NS3 helicase domain is considered a virulence determinant [52,91]. In particular, increased avian virulence due to the point mutation NS3-T249P has been reported in American crows (Corvus brachyrhynchos) and in site-directed mutagenesis experiments [73,[92][93][94]. In support of our analysis, the Italian strain MW627239 is not characterized by the NS3-T249P point mutation. A threonine residue was observed at the 249 position in the strain MW627239, as well as in JF719065 and MW835362, circulating in Italy in 2008 (jay) and 2011 (owl), and in JF719069 and MT863559, circulating in Spain (horse) and France (human) in 2010 and 2015, respectively. Furthermore, the amino acid valine at the residue 159 of the E protein is considered a determinant of WNV neurovirulence, influencing viral replication and pathogenesis, and being involved in WNV infection and T-cell infiltration in the brain [75]. This amino acid is observed in the isolate NC009942, circulating in the USA in 1999, but not in the Italian and European strains, all characterized by isoleucine at this residue position. This point mutation might also help explain the low pathogenesis observed among birds in Italy and, more generally, in Europe. It is likely that the WNV pathogenicity is the result of a complex series of events which involve the virus, the vectors, and the hosts. Further studies correlated with the WNV genotype and phenotype may help in understanding the mechanisms underlying WNV clinical signs in birds, and the emergence of new pathogenic phenotypes.

Conclusions
In recent years, WNV-L2 has been, by far, the most frequent lineage detected in Italy, whereas WNV-L1 was detected only occasionally. Since 2008, about 1500 wild birds and 5000 resident birds belonging to target species (carrion crow, magpie, Eurasian jay) have been annually tested by real time RT-PCR to monitor the circulation of WNV L1 and L2 strains in Italy. Among them, 326 wild birds and 804 target species were found positive to WNV (https://westnile.izs.it/j6_wnd/home, 16 December 2021). Concerning WNV-L1, it was last detected in a sparrow hawk (Accipiter nisus) and in two carrion crows in the Sardinia region during the 2016 vector season.
Serological analysis conducted in Italy on humans and horses between 2008 and 2020 identified anti-WNV-IgM in 1189 persons and 1196 horses. No evidence of WNV circulation was detected in the Campania region [95] (https://westnile.izs.it/j6_wnd/ home, 16 December 2021). The detection of the WNV-L1 strain in two wild birds described in this study emphasizes the importance of having in place an efficient surveillance system, and, in particular, the early warning function played by some avian species in detecting WNV circulation.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/v14010064/s1, File S1: WNV Lineage 1 Sequence Alignment.  Data Availability Statement: Sequence data are available via NCBI. The accession numbers for the sequences used can be found in Table 1.